Ligand gold bonding

ABSTRACT

Gold sol coated with alkanethiols and alkanethiol derivatives, which provide groups on the sol available for the linking of binding moieties such as antibodies, antigens or ligands to the gold sol. Di- and tri-thiol compounds bound to gold sol also facilitate the adsorption of antibodies, antigens or ligands to the sol. The coating process, and test kits incorporating the coated sols are also included.

BACKGROUND

This invention relates to gold sol coated with alkanethiols andalkanethiol derivatives to provide groups on the sol available for thebinding or linking of binding moieties such as antibodies, antigens, orligands to the gold sol. In addition, the use of di- and tri-thiolcompounds bound to gold sol facilitate the passive adsorption of thesebinding moieties. This invention also relates to gold sol coated withthiolated binding moieties, including antigens, antibodies, or carriermolecules, which can be attached to relevant ligands. Also included isthe process for coating the gold sols with such thiol compounds, the useof coated sols in immunological and immunocytological diagnostic testsand test kits incorporating such coated gold sols.

Test methods for the diagnosis of various diseases are constantly beingimproved. Currently, immunological methods are among the most sensitivemethods used to detect the presence of antigens or antibodies insamples. These assays are well known to those skilled in the art ofimmunodiagnostics. Generally, an immunological assay consists of anassay wherein a monoclonal or polyclonal antibody is used to capture anantigen in the sample and a second antibody containing a label, such asa fluorescent compound or an enzyme, immunochemically reacts with theantigen-antibody complex. The resulting labelledantibody-antigen-antibody complex is detected. Variations on this basicassay are common, such as the use of only one reactive antibody in thetest, the competitive inhibition method, or the use of particles aslabels that allow an agglutination reaction to be read. Another variantis the use of microparticles coated with either an antigen or anantibody, which after formation of a complex with the analyte and asecond appropriately labeled binding partner, gives a positive reaction.

Gold sol microparticles are used in an assay method known as a solparticle immunoassay (SPIA). In this assay, a solution containing thegold sol coated with an appropriate binding partner, either an antibodyor an antigen, is reacted with a sample to bind to its binding partner.In this process, complexes are formed that can be detected, usually dueto their change in color.

Uncoated gold sol particles, and other colloids, will undergoagglomeration when exposed to low concentrations of salt, and quicklyprecipitate out of solution. Therefore, the coating of gold sol with anappropriate binding partner serves two functions. The first is toprovide appropriate immunological binding activity and the second is toprotect against agglomeration, which would of necessity occur in buffersdesigned to optimize for immunological reactions. Since not all proteinsor polymers will protect the gold sol completely from salt-inducedagglomeration, an overcoating step is usually performed with a proteinor polymer that is known to be capable of completely protecting the goldsol from salt-induced agglomeration. Such an overcoating step is a wellknown practice in adsorbing antibodies and antigens to plasticsubstrates and has been shown to increase the stability of the coatedmaterial.

The overcoating also serves to reduce nonspecific interaction of thegold sol with sample components. This nonspecific interaction is asignificant problem when using antibody coated latex particles indiagnostic assays, and is probably a manifestation of the nonspecificserum interference observed in many, if not all, immunoassays,regardless of format. In some cases it is permissible to change theovercoating protein or polymer to minimize interference in specificsystems. Additives to the sol medium such as guanidine hydrochloride orurea are useful. Occasionally, "non-specific" interference can bepinpointed to serum heterophile activity and is eliminated by theaddition of whole animal serum.

A serious drawback in the passive adsorption of the desired bindingpartner to gold colloid has been that such direct coating is oftenunsuccessful. The physico-chemical mechanisms of passive polymeradsorption to colloids in general is a poorly understood process.Passive adsorption of antibodies to gold sol may result in a coated solwhich is poorly protected from salt-induced agglomeration and whichcannot be further protected by overcoating. It may also result in acoated sol with poor immunological activity, presumably due to theincorrect orientation of adsorbed antibody, or it may result in theantibody-induced agglomeration of the sol itself. Finally, the bindingpartner of choice may simply not bind to the gold sol. The net result ofthese problems is that few biological reagents useful in otherdiagnostic formats can be used in the production of gold colloidreagents of diagnostic quality.

What is needed in the art is a method for covalently attaching bindingpartners, proteins, carbohydrates or ligands, to the gold sol so thatthe uncertainties of the passive adsorption characteristics or thenecessity of making binding partner and "good coating" carrierconjugates for passive adsorption may be eliminated. In particular, sucha sol would be significantly more useful if it were refractive tosalt-induced agglomeration even in the absence of a coated bindingpartner. The ability to change the physico-chemical surface propertiesof the sol would, at the same time, make it possible to minimize thesometimes undefined sample-sol interactions responsible for non-specificinterference in immunochemical diagnostic assays and background problemsin immunocytochemical assays.

What is also needed is the ability to facilitate the passive adsorptionof biological polymers to gold sols. Thiolation of antibodies andpolymers can increase their ability to bind to gold sols as evidenced byincreased resistance to salt-induced agglomeration. Such a capabilitymay prove useful for those antibodies which bind well to gold sols, butlose significant amounts of their activity in doing so as well as forantibodies which simply do not bind the gold sol in an underivatizedstate. An alternative method of changing the physico-chemicalcharacteristics of the sol surface in order to facilitate antibodybinding is the coating of the sol with di-thiol or tri-thiol compounds.Such an intermediate coating can significantly change the passiveadsorption properties of antibodies to the coated sol.

SUMMARY OF THE INVENTION

This invention provides a process for coating microparticles of gold solwith alkanethiols, alkanethiol derivatives, and di- and tri-thiolcompounds. Gold sols coated with alkanethiols or their derivatives areresistant to salt-induced agglomeration and contain chemical moietiesfor covalent polymer or ligand attachment. A particularhydrophobic-hydrophilic balance of the sol surface is obtained andnonspecific interactions of the sol with proteins generally areminimized. In particular, chemical groups distal to the n-alkane thiolmoiety, such as methyl, hydroxyl, carboxyl, amino, sulfhydryl orcarbonyl, are solvent exposed and serve as covalent attachment sites andmay be hydrophobic-hydrophilic balance sites.

Coating gold sol with small molecular weight di- and tri-thiol compoundsdoes not protect the sol from salt-induced agglomeration, but changesthe physico-chemical nature of the coated sol surface and facilitatesthe passive adsorption of antibody molecules.

Also included in the present invention is the coated gold sol. Theinvention also includes coated gold sol particles additionally bound tobinding moieties such as antibodies or antigens and diagnostic kitscontaining said gold sol particles.

Lastly, the invention includes coated gold sols to which bindingmoieties are attached by adsorption, and uncoated gold sol whereadsorption is facilitated through the thiolation of the binding moiety.

DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the relative resistance to salt-induced gold solagglomeration conferred by coating the gold sol with anti-gp160 antibody(GC-143) at various concentrations and pH values.

FIG. 2 demonstrates the relative resistance to salt-induced gold solagglomeration conferred by coating a TTC coated gold sol with anti-gp160antibody (GC-143) at various concentrations and pH values.

FIG. 3 demonstrates the ability of GC-143 to cause spontaneousagglomeration of a gold sol in the absence of any added salt at variousconcentrations and pH levels.

FIG. 4 demonstrates the ability of GC-143 to cause spontaneousagglomeration of a TTC coated gold sol in the absence of any added saltat various concentrations and pH levels.

FIG. 5 shows the immunological activity and specificity of GC-143adsorbed on a gold sol in a SPIA assay.

FIG. 6 shows the immunological activity and specificity of GC-143adsorbed on a TTC coated gold sol in a SPIA assay.

FIG. 7 demonstrates the ability of an underivatized monoclonal anti-HIVp24 antibody adsorbed to gold sol to protect the sol from salt-inducedagglomeration.

FIG. 8 demonstrates the ability of thiolated monoclonal anti-HIV p24antibody adsorbed to gold sol to protect the sol from salt-inducedagglomeration.

DETAILED DESCRIPTION OF THE INVENTION

The process of coating microparticles consisting of gold sol to providechemical linking groups to facilitate the further attachment of othergroups is the basis of the present invention. Microparticles in theinvention are generally defined as colloidal gold sol that range in sizefrom about 20 nm to about 200 nm or more. The preferred range ofmicroparticle is from about 60 to about 80 nm. The most preferred sizeis about 65 to 75 nm. Some of the coating chemicals used, such asalkanethiols and their derivatives and mixtures thereof also protect thegold sols from salt-induced agglomeration and may produce a particularhydrophobic-hydrophilic balance of the sol surface so that nonspecificinteractions of the surface with extraneous proteins are minimized.

Gold sol was produced by the hydroxylamine-mediated reduction ofchloroauric acid in water onto seed gold particles. This procedure isdescribed in the literature. (Turkevich, J. et al., Discussions of theFaraday Society, No. 11, p. 55-74 (1951)). Generally, chloroauric acidtrihydrate dissolved in water is added to deionized water, to which isthen added hydroxylamine hydrochloride. Seed gold particles are addedand stirred. A small amount of acetone is added, and the mixture isstirred. K₂ CO₃ is added until the pH reaches 7.0. The gold sol rangesin size from about 20 nm to about 150 nm in diameter depending on theamounts of gold particle added and the amount of chlorauric acid used,and is now ready to be coated with alkanethiols or thiol derivatives.

Alkanethiols, their derivatives, and di- and tri-thiol compounds are thepreferred compounds used to coat the gold sol particle and to providechemical moieties or linking groups for covalent polymer or ligandattachment to the gold sol or for the modification of thehydrophilic/hydrophobic balance. The more preferred are then-alkanethiols, while the most preferred compounds are alkanethiols ofthe formula CH₃ (CH₂)_(n) SH, where n equals 9 to 23. Derivatives of thealkanethiols are generally of the formula RCH₂ (CH₂)_(n) SH wherein R isOH, COOH, CHO, SH and NH₂, and n is 9 to 23. The di- and tri-thiolcompounds are generally compounds of low molecular weight. Some of themhave been recognized as heavy metal chelators. Two examples are2,3-dimercaptosuccinic acid and trithiocyanuric acid.

To coat a sol with an n-alkanethiol, 0.1M of the alkanethiol and 1%Tween-20^(R) (ICI Americas registered trademark, polyoxyethylenesorbitanmonolaurate) in alcohol, preferably methanol, is freshly prepared.Approximately 1 ml of this mixture is added to approximately 100 ml ofthe prepared gold sol, and is stirred. The resulting mixture is allowedto set at room temperature for approximately 2 hours. Lowerconcentrations of the coating compound will effectively coat gold solbut the optimal coating times must be found empirically for each case.

The sol mixture is washed into an approximately 1 mM3-[N-Morpholino]-2-hydroxypropane sulfonic acid (MOPSO), pH 7 buffer bycentrifugation at 2000×g for approximately 15 minutes at roomtemperature, and can be stored at 4° C. until use.

The above is an example of coating gold sol with an n-alkanethiolcompound. This coating procedure is modified for the attachment ofmixtures of n-alkanethiols and n-alkanethiol derivatives containinglinking groups for the chemical conjugation of ligands, or polymericcompounds. For example, the inclusion of an hydroxy-n-alkanethiol to thecoating mixture increases the hydrophilicity of the sol surface anddecreases the non-specific interaction of the sol with serum proteins.The inclusion of a carboxyl-n-alkanethiol at a level of 10% of totalthiol compounds present is sufficient to produce a sol with the desireddensity of conjugation moieties. The carbon chain length of then-alkanethiol in these mixtures can be 2-4 carbons less than the carbonchain length of the n-alkanethiol derivatives in order to better exposethe conjugation moieties to reduce steric hindrance associated withreaction of macromolecules with the sol surface.

The coating solution of n-alkanethiol, n-alkanethiol derivative ormixtures of the two groups is generally made in alcohol, preferablymethanol, with 1% Tween-20^(R). The total concentration of all the thiolcompounds is generally no greater than 0.10M. In some cases, the optimalpH for coating thiol compounds is not 7. For example, in the case oftrithiocyanuric acid coated sols, the sol is adjusted to pH 6 before theaddition of the thiol compound to optimize the degree of resistance tosalt-induced agglomeration conferred by a particular thiol compoundmixture.

The sols are generally allowed to coat in the presence of the thiolmixture for 2 hours, but optimal incubation times are determinedempirically for each case. This is accomplished by testing the sols fortheir resistance to salt-induced agglomeration after incubation periodsof varying length using a spectrophotometric assay. Gold sols have acharacteristic violet color, which is exceedingly sensitive to thediameter of the sol particles. If sol particle agglomeration takesplace, the effective "size" of the sol is greatly increased and thecharacteristic absorption maximum shifts from 540 nm, which is violet,to higher wavelengths, with a blue to colorless sol color.

In practice, 1 ml of the coated sol aliquot is dispensed into a glasstube. One hundred microliters of 10% NaCl are added to the sol aliquotwith mixing. The mixture is allowed to set at ambient temperature for 10minutes and its absorbance at 540 nm is compared to an identical solaliquot to which 100 microliters of water have been added. A testaliquot that retains 90% or greater of the absorbance of the controlaliquot is considered to be resistant to salt-induced agglomeration.

Compounds tested are shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                                             Relative Degree                                                               of Protection                                                                 From Salt-Induced                                        Compound(s)          Agglomeration*                                           ______________________________________                                        n-hexadecanethiol    97%                                                      n-dodecanethiol      93%                                                      n-hexadecane         25%                                                      1-hexadecanoic acid  26%                                                      12-mercapto-1-dodecanoic acid                                                                      46%                                                      11-mercapto-1-undecanol                                                                            .sup. --.sup.1                                           2,3-dimercapto succinic acid                                                                       41%                                                      2,5-dimercapto-1,3,4-thiadiazole                                                                   .sup. 94%.sup.2                                          3-amino-5-mercapto-1,2,4-triazole                                                                  <30%                                                     trithiocyanuric acid <30%                                                     n-dodecanethiol:                                                              12-mercaptododecanoic acid                                                    (1:1).sup.3          74%                                                      (4:1)                76%                                                      (9:1)                90%                                                      n-decanethiol:11-mercapto-1-undecanol:                                        12 mercapto dodecanoic acid                                                   (8:1:1)              100%                                                     (6:3:1)              83%                                                      ______________________________________                                         *expressed as % optical density at 540 nm of control sol aliquot with no      added salt.                                                                   .sup.1 the sol was spontaneously agglomerated in the presence of the          coating compound                                                              .sup.2 even though the sol was protected from saltinduced agglomeration,      after harvesting by centrifugation the sol was no longer salt protected.      .sup.3 mole ratios of compounds used in coating mixtures                 

The compounds found to be most successful in coating the gold sol andprotecting it from salt-induced agglomeration were n-alkanethiols.N-alkanethiol derivatives such as 12-mercapto-1-dodecanoic acid wereineffective by themselves in conferring resistance to salt-inducedagglomeration, but sols coated with mixtures of n-alkanethiols and theirderivatives did confer complete protection from salt-inducedagglomeration and contributed the other properties discussed above.

The coated gold sol containing chemically conjugatable groups is thenbound to binding moieties such as proteins, carbohydrates, antibodies,antigens, polymers, monomers or ligands using carbodiimide chemistry bywell known methods. Binding moieties include any groups that are capableof being adsorbed or covalently attached to the coated gold sol. In somecases, carbodiimide chemistry is also used to conjugate a linkermolecule such as 6-amino caproic acid to the sol prior to the indirectconjugation of the binding partner in order to relieve stericconstraints on the binding partner and aid in its recognition by and ofits complementary binding partner. In order to conjugate the bindingpartner or the linker molecule to the coated sol, the sol is incubatedat ambient temperature in the presence of the substance to be coupled,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC), andN-hydroxysulfosuccinimide(SNHS), in a 10-25 mM buffer at pH 7.0 for 2hours. In some cases, sequential aliquots of EDC are added during theconjugation reaction in order to compensate for the hydrolysis of thiscompound in aqueous media.

The choice of potentially useful conjugation chemistries is determinedby the chemical moiety on the coated sol as well as by the type ofmoiety introduced on any given linker molecule. For example, aminogroups on the sol-bound alkanethiol derivatives or attached linkermolecules could be conjugated to polymers using homo- orhetero-bifunctional N-hydroxysuccinimide ester crosslinkers. Likewise,sulfhydryl groups on sol-bound alkanethiol derivatives or linkermolecules could be conjugated to polymers using homo- orhetero-bifunctional maleimide crosslinkers. The range of possibleconjugation schemes is very similar, if not identical to, the range ofchemical schemes currently used in the preparation of diagnosticimmunoconjugates. Classical crosslinking reagents compatible with themoieties on the sol, the alkanethiol derivatives or the chemicalcrosslinkers are needed to attach the appropriate binding partner to thesol.

Immunoassays using the sol particle immunoassay (SPIA) method areperformed as described in U.S. Pat. No. 4,313,734 to J. Leuvering.Briefly, gold sol coated with a binding moiety is incubated in thepresence of a test sample containing the opposite binding partner. Ifthe opposite binding partner is present in the sample, cross linking ofthe coated sol will result and the absorbance at the absorption maximumof single gold sol particles will decrease dramatically. The adsorptionproperties of polymeric compounds to gold sols may be facilitated by thethiolation of the binding partner.

Both polyclonal and monoclonal antibodies were thiolated by reactionwith N-succinimidyl S-acetylthioacetate (SATA). The resulting protectedsulfhydryl groups were exposed by reaction of the proteins withhydroxylamine hydrochloride. This is a well known chemical procedure forintroducing sulfhydryl groups into protein. The thiolated proteins werethen adsorbed to gold sols at pH's 6, 7, 8 and 9 at proteinconcentrations ranging from 5 μg/ml to 100 μg/ml. The degree ofresistance to salt-induced agglomeration was determined as describedabove. Thiolation of antibodies by this method significantly improvedtheir ability to protect gold sol from salt-induced agglomeration overnon-thiolated antibodies, as shown in FIGS. 7 and 8, and described inExample 12.

An alternative method of facilitating the adsorption of antibodies togold sol is the inclusion of an intermediate coating between the goldsol and the binding partner in order to change the physico-chemicalproperties of the sol surface such that adsorption of the antibodies isincreased. Di- and tri-thiols are used for such a purpose.Trithiocyanuric acid (TTC) is an example of such a compound. Gold solwas coated with TTC in a procedure similar to that described above forthe n-alkanethiol compounds but with different concentrations of TTC andTween-20™. These concentrations were determined empirically. The abilityof antibody to confer resistance to salt-induced agglomeration of thewashed sol was determined after incubation of the antibody and thecoated sol at various pH's and protein concentrations in the previousparagraph.

The coated microparticles of this invention may be covalently bound toantigen or antibodies and sold in a test kit for use inimmunodiagnostics. The antigen or antibody will be specific for theopposite binding partner sought to be detected, and the other componentsof the kits may include diluents, buffers, labeling reagents andlaboratory equipment that will also be specific for the particular testto be performed.

The following examples are given to more fully explain, but not to limitthe invention. Modifications of the invention will be obvious to thoseskilled in the art and are also considered as part of this invention.

EXAMPLES Example 1 Preparation of Gold Sol

For preparation of the seed sol used in the final gold sol preparation,10 ml of 1% HAuCl₄ trihydrate was added to 900 ml of water and stirredwell. 10 ml of 1% sodium citrate was added to the mixture and stirredwell. With vigorous stirring, 10 ml of 0.075% sodium borohydride wasadded (made by dissolving sodium borohydride in the sodium citratesolution) and stirred well for 5 minutes. The mixture was filteredthrough a 0.22 micron filter and stored at 4° C. The sol should age atleast 24 hours before use.

For preparation of the final gold sol, 8 ml of 2% HAuCl₄ trihydrate (inwater) was added to 900 ml of water. With continuous stirring, 4 ml offreshly prepared 5% hydroxylamine hydrochloride was added (in water).Immediately thereafter, 22.33 microliters of seed sol was added andstirred vigorously for 30 minutes. 1 microliter of acetone was added foreach milliliter of sol produced and stirring continued for 10 minutes.0.2M K₂ KCO₃ was added until the desired pH was obtained.

The appropriate volume of seed sol added was determined by the followingrelationship: Volume of seed required for desired sol size=volume ofseed required for sol of given size times the cube root of (Diameter ofsol obtained from a given amount of seed sol divided by diameter of thedesired particle).

Example 2 Coating of Gold Sol with n-Alkanethiol

Gold sol was prepared as described in Example 1. A solution of 0.1M1-dodecanethiol in methanol and 1% Tween-20™ was freshly prepared. Forevery 100 ml of gold sol to be coated, 1 ml of the thiol-methanolsolution was added dropwise with stirring to the gold sol. The mixturewas allowed to set at ambient temperature with occasional swirling for 2hours. The coated gold sol was recovered by centrifugation at 2000×g for15 minutes at ambient temperature with subsequent resuspension in 1 mMMOPSO, pH 7.0. This centrifugation and resuspension was repeated 3times. The resultant sol was resuspended in the same buffer and storedat 4° C. prior to use.

Example 3 A n-Alkanethiol and n-Alkanethiol Derivative Mixture CoatedGold Sol

Gold sol was prepared as described in Example 1. A solution of 0.09M1-dodecanethiol, 0.01M 12-mercapto-1-dodecanoic acid in methanol and 1%Tween-20™ was freshly prepared. For every 100 ml of gold sol to becoated, one ml of the thiol mixture-methanol solution was added dropwisewith stirring to the gold sol. The mixture was allowed to set at ambienttemperature with occasional swirling for 2 hours. The coated gold solwas recovered in a manner identical to the previous example.

Example 4 n-Alkanethiol Derivative Gold Sol Coating and Subsequent Lackof Protection from Salt-Induced Agglomeration

Gold sol was prepared as described in Example 1 and adjusted to ph 7with K₂ CO₃. A solution of 0.10M 13-mercapto-1-dodecanoic acid and 1%Tween-20™ in methanol was freshly prepared. For every 100 ml of gold solto be coated, 1 ml of the thiol-methanol solution was added dropwisewith stirring to the gold sol. The mixture was allowed to set at ambienttemperature with occasional swirling for 1 hour. To duplicate 1 mlaliquots of the coated sol were added either 100 μl of water or 10%NaCl. The aliquot to which water was added was considered the control.The aliquot to which 10% NaCl was added had lost 54% of its opticaldensity after 10 minutes. This is interpreted as evidence that the goldsol was not protected from salt-induced agglomeration by being coatedwith the 12-mercapto-1-dodecanoic acid.

Example 5 Preparation of a Gold Sol Coated with a Mixture ofn-Alkanethiol and n-Alkanethiol Derivatives

Gold sol was prepared as described in Example 1 and adjusted to pH 7with K₂ CO₃. A solution of 0.004M N-decanethiol, 0.0005M11-mercapto-1-undecanol, 0.0005M 12-mercapto-1-dodecanoic acid, 0.08MNaCl and 1% Tween-20™ in methanol was freshly prepared. For every 100 mlof sol to be coated, 1 ml of the thiol-thiol derivative solution wasadded dropwise with stirring and the mixture was allowed to setovernight at ambient temperature. The coated sol was assayed forprotection from salt-induced agglomeration as in Example 4.

The coated sol retained 100% of its optical density at 540 nm after theaddition of 10% NaCl. This is believed to show that the coated sol isfully protected from salt-induced agglomeration.

Example 6 Bovine Serum Albumin (BSA) Conjugation to Coated Gold Sol

Four ml of the coated gold sol described in Example 3 with an opticaldensity at 540 nm of 25.0 was mixed with 4.3 ml of 10 mM MOPSO, pH 7.0.To this mixture was added sequentially with stirring, 0.2 ml of 25 mg/mlBSA, 1.0 ml of 30 nm SNHS, and 0.5 ml of 0.1M EDC. The BSA, SNHS and EDSwere prepared in 10 mM MOPSO, pH 7.0. The reaction was rocked for 2hours at ambient temperature. The reaction was quenched by an additionalhour of rocking after the addition of 1 ml of 1.0M ethanolamine(adjusted to pH 7.0 with HCl), pH 7.0 in 10 mM MOPSO. An equal volume of10 mM MOPSO, 0.15M NaCl, 0.5% Tween-20™, 1.0 mg/ml casein, at pH 7.0 wasadded to the reaction mixture and the mixture was allowed to set for 1hour. This blocking or overcoating step protects the unreacted bareareas of the gold sol surface from nonspecific interaction with otherbiopolymers. The BSA conjugated coated sol was recovered bycentrifugation at 2000×g at ambient temperature for 15 minutes andsubsequent resuspension in the same buffer. This centrifugation and washwas repeated 3 times. The sol was centrifuged as above and resuspendedin 10 mM MOPSO, pH 7.0. This step was repeated twice. The BSA conjugatedcoated sol was stored at 4° C. prior to use.

Example 7 SPIA of BSA Conjugated Gold Sol

The BSA conjugated gold sol from Example 5 was diluted in 0.1M MOPSO,0.15M NaCl, 1.0% polyethylene glycol 8000, pH 7.0 to obtain an opticaldensity at 540 nm of 1.0. Polyclonal anti-BSA antibody and normal rabbitimmunoglobulin(IgG) were diluted in 10 mM MOPSO, pH 7.0 to 12microliters/ml. To separate 1 ml aliquots of BSA conjugated sol wereadded 100 microliters of the diluted anti-BSA or normal rabbit IgG, or10 mM MOPSO buffer with mixing. The mixtures were transferred tocuvettes and the optical densities at 540 nm were monitored over timeand shown in Table 2.

The results in Table 2 demonstrate that there is immunologicallyrecognizable BSA on the surface of the sol and that the decrease inoptical density is not due to non-specific interaction of rabbit serumwith the sol.

Example 8 SPIA of BSA Negative Control Conjugated Gold Sol

A negative control BSA conjugated coated sol was prepared exactly as inExample 5 above except that no EDC was added to the conjugationreaction. An identical volume of 10 mM MOPSO, pH 7.0 buffer was includedinstead. The purpose of this sol was to demonstrate in SPIA assays thatthe BSA activity observed was covalent in nature. Comparison of resultsin Tables 2 and 3 demonstrate that although a significant portion of theBSA activity on these sols may be passively adsorbed, an equallysignificant portion is covalently bound.

                  TABLE 2                                                         ______________________________________                                                  BSA CONJUGATED COATED SOL                                           SAMPLE    DECREASE IN O.D. 540 nm AT 60'                                      ______________________________________                                        Anti-BSA  0.401                                                               Rabbit IgG                                                                              0.029                                                               Buffer    0.031                                                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                BSA NEGATIVE CONTROL CONJUGATED                                       SAMPLE  GOLD SOL DECREASE IN O.D. 540 nm AT 60'                               ______________________________________                                        Anti-BSA                                                                              0.238                                                                 Rabbit IgG                                                                            0.063                                                                 Buffer  0.050                                                                 ______________________________________                                    

Example 9 Improvement in Coating Properties of Antibody on TTC CoatedGold Sol

Gold sol was prepared as described in Example 1, except that its pH wasadjusted to 6.0 with K₂ CO₃. A saturated solution of TTC in methanol wasfreshly prepared. A solution of 10% Tween-20™ in methanol was addeduntil the final Tween-20™ concentration was 2.5%. Four ml of thissolution was added to 200 ml of the above sol with stirring and themixture was allowed to set at ambient temperature for 2 hours withoccasional swirling. The coated sol was harvested as in Example 2.Separate aliquots of the coated sol were diluted to an optical densityof 2.0 and simultaneously adjusted to pH 6, 7, 8 and 9 with 10 mM(2-[N-morpholino]ethanesulfonic acid) (MES), 10 mM MOPSO, 10 mM(N-[2-hydroxethyl]-piperazine-N-[3-propanesulfonic acid]) (EPPS), and 10mM (2-[N-cyclohexylamino]-ethanesulfonic acid) (CHES), respectively. Inparallel, a gold sol was prepared as in Example 1 and separate aliquotswere adjusted to pH 6, 7, 8 and 9 with K₂ CO₃. A purified human anti-HIVpolyclonal antibody was diluted to 1.0, 0.5, 0.25, 0.10 and 0.05 mg/mlin each of the above buffers. To parallel 1.0 ml aliquots of each of theabove sols and each pH was added 100 μl of antibody solution at thecorresponding pH with mixing. The mixtures were allowed to set atambient temperature for 30 minutes. One hundred μl of 10% NaCl was addedto each sol mixture with agitation. After an additional 10 minutes, theoptical density at 540 nm of each mixture was measured, as shown inFIGS. 1-4.

The adsorption of the antibody on the TTC coated sol resulted in abroader pH range at which protection from salt-induced agglomerationoccurred. Also, protection from salt-induced agglomeration took place atlower antibody concentrations on the TTC coated sol. In the absence ofNaCl addition, the antibody caused spontaneous agglomeration of theuncoated sol. The antibody-induced spontaneous agglomeration was muchless apparent on the TTC coated sol.

Collectively, these observations show that the adsorption of thisantibody on TTC coated gold sol is greater than that seen on uncoatedsol.

Example 10 Improvement in SPIA Activity of Antibody Adsorbed on TTCCoated Gold Sol

One hundred ml of gold sol was prepared as in Example 1 and the pH wasadjusted to 9.0 Two and one-half milliliters of 2 mg/ml of the humananti-HIV (GC-143) in 10 mM CHES, pH 9.0 was added with stirring and themixture was allowed to set at ambient temperature for approximately 2hours with occasional swirling. Five ml of 1 mg/ml nonfat dry milk wasadded to the mixture with stirring and the mixture was allowed to set atambient temperature for an additional hour. The coated sol wascentrifuged at 2000×g for 15 minutes at ambient temperature andresuspended in 10 mM CHES, 0.5% BSA, 300 mM mannitol, 0.01% sodiumazide, at pH 9.0. The centrifugation and resuspension step was repeated3 times. The sol was finally resuspended in the same buffer and storedat 4° C. prior to use. A TTC coated sol prepared as in Example 9 wasdiluted with 10 mM CHES at pH 9.0 to obtain 60 ml of sol with an opticaldensity of 2.0, the same approximate optical density of the gold solprepared in Example 1. Six ml of 0.5 mg/ml human anti-HIV dissolved in10 mM CHES, pH 9.0 was added with stirring and the mixture was allowedto set at ambient temperature for 1 hour, then overnight at 4° C. Six mlof 1 mg/ml nonfat dry milk was added with stirring and the mixture wasallowed to set at ambient temperature for 1 hour. The coated sol washarvested as above and stored identically.

Aliquots of each sol were diluted to an optical density at 540 nm of 2.0with 0.1M MOPSO, 0.15M NaCl, 1% PEG 8000, 0.25% BSA, at pH 7.0. Toreplicate aliquots of each diluted sol were added a) one hundred μlbuffer, b) one hundred μl of 10 μg/ml HIV gp160, or c) one hundred μl ofa mixture of 5 μg/ml HIV gp160 and 5 μg/ml human anti-HIV which had beenpre-incubated for 90 minutes at ambient temperature, as shown in FIGS. 5and 6.

It is apparent that the GC-143 TTC coated sol is more immunologicallyactive than its uncoated sol counterpart and that its use in the SPIAassay results in a more sensitive assay. Therefore, by functionalcriteria as well, the GC-143 TTC coated sol is also better than theuncoated sol counterpart.

Example 11 Thiolation of Monoclonal Anti-HIV p24 usingN-succinimidyl-S-acetylthioacetate (SATA)

One ml of monoclonal anti-HIV p24 at 1.46 mg/ml in 50 mM NaPO4, 1 mMethylenediamine tetraacetic acid (EDTA), pH 7.5 was mixed with 0.02 mlof 5 mg/ml SATA in dimethylsulfoxide (DMSO) and allowed to react atambient temperature for 2 hours. The reacted antibody was separated fromthe reaction components by gel filtration through a desalting column inthe same buffer. One quarter mg of the reacted gel filtered antibody wasdiluted to 1 ml with the same buffer and 100 μl of 0.05M NaPO4, 25 mMEDTA, 0.5M NH2OH.HCl, at pH 7.5 was added with mixing. The reaction wasallowed to set for 1.5 hours at ambient temperature. The deprotectedantibody was gel filtered through a desalting column into the originalbuffer to remove unreacted reagents.

Example 12 Improvement of Coating Properties of Thiolated MonoclonalAnti-HIV p24

Gold sol was prepared as described in Example 1 except that separatealiquots of the sol were adjusted to pH 6, 7, 8 and 9 with K₂ CO₃. Inparallel, separate aliquots of the underivatized and derivatizedmonoclonal anti-HIV p24 (see Example 11) were adjusted to 250, 200, 150,100 and 50 μg/ml. For each of the above, the relative ability ofderivatized and underivatized antibody to protect the gold sol fromsalt-induced agglomeration was made exactly as described in Example 9(see FIGS. 7 and 8).

It is apparent from FIGS. 7 and 8 that the thiolation of this monoclonalantibody resulted in improved coating characteristics as judged by a)the fact that the derivatized antibody did not spontaneously agglomeratethe sol, b) the decreased dependence of antibody protection against solagglomeration on pH, and c) the lower relative amounts of derivatizedantibody required to protect from salt-induced sol agglomeration in thepresence of salt as compared to the underivatized antibody sol.

We claim:
 1. Microparticles comprising gold sol coated with at least onecompound selected from the group consisting of alkanethiol, di-thiol andtri-triol compounds.
 2. Microparticles according to claim 1, whereinsaid compound is an alkanethiol.
 3. Microparticles according to claim 1,wherein said alkanethiol is a n-alkanethiol of the formula CH₃ (CH₂)_(n)SH, and n equals a whole number from 9 to 23 inclusive. 4.Microparticles according to claim 1, wherein said compound is a di-thiolcompound.
 5. Microparticles according to claim 1, wherein said compoundis a tri-thiol compound.
 6. Microparticles according to claim 1, whereinsaid gold sol has a diameter of from about 10 nm to about 150 nm. 7.Microparticles according to claim 1 which are resistant to salt-inducedagglomeration, whereby no more than 10% of the optical density at 540 nmof a suspension of said sol is lost after 10 minutes in the presence of1.0% NaCl.
 8. Microparticles according to claim 1, wherein saidmicroparticles are covalently linked to a binding moiety. 9.Microparticles according to claim 8, wherein the binding moiety isselected from the group consisting of proteins, carbohydrates, antigensand ligands.
 10. Microparticles according to claim 8, wherein thebinding moiety is a polymer or monomer.
 11. Microparticles according toclaim 1, additionally comprising a bridging compound which is covalentlyattached to said compound and to which is covalently linked a bindingmoiety.
 12. Microparticles according to claim 1, wherein additionally abinding moiety is passively adsorbed.
 13. A diagnostic kit for use inimmunoassays comprising microparticles according to claim 1, and afurther solution.
 14. Microparticles comprising gold sol coated with acompound of the formula RCH₂ (CH₂)_(n) SH, where R is selected from thegroup consisting of OH, COOH, CHO and NH₂, and n is 9 to
 23. 15.Microparticles comprising gold sol coated with a compound of the formulaSHCH₂ (CH₂)_(n) SH, and n is 9 to 23.